REVIEW Open Access

Effects of mesenchymal stromal cell-conditioned media on measures of lungstructure and function: a systematic reviewand meta-analysis of preclinical studiesAlvaro Moreira1* , Rija Naqvi1, Kristen Hall1, Chimobi Emukah1, John Martinez1, Axel Moreira2, Evan Dittmar1,Sarah Zoretic1, Mary Evans1, Delanie Moses1 and Shamimunisa Mustafa1

Abstract

Background: Lung disease is a leading cause of morbidity and mortality. A breach in the lung alveolar-epithelialbarrier and impairment in lung function are hallmarks of acute and chronic pulmonary illness. This review is parttwo of our previous work. In part 1, we demonstrated that CdM is as effective as MSCs in modulating inflammation.Herein, we investigated the effects of mesenchymal stromal cell (MSC)-conditioned media (CdM) on (i) lungarchitecture/function in animal models mimicking human lung disease, and (ii) performed a head-to-headcomparison of CdM to MSCs.

Methods: Adhering to the animal Systematic Review Centre for Laboratory animal Experimentation protocol, weconducted a search of English articles in five medical databases. Two independent investigators collectedinformation regarding lung: alveolarization, vasculogenesis, permeability, histologic injury, compliance, andmeasures of right ventricular hypertrophy and right pulmonary pressure. Meta-analysis was performed to generaterandom effect size using standardized mean difference with 95% confidence interval.

Results: A total of 29 studies met inclusion. Lung diseases included bronchopulmonary dysplasia, asthma,pulmonary hypertension, acute respiratory distress syndrome, chronic obstructive pulmonary disease, andpulmonary fibrosis. CdM improved all measures of lung structure and function. Moreover, no statistical differencewas observed in any of the lung measures between MSCs and CdM.

Conclusions: In this meta-analysis of animal models recapitulating human lung disease, CdM improved lungstructure and function and had an effect size comparable to MSCs.

Keywords: Conditioned media, Mesenchymal stem cell, Lung disease, Animal, Review

© The Author(s). 2020 Open Access This article is licensed under a Creative Commons Attribution 4.0 International License,which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you giveappropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate ifchanges were made. The images or other third party material in this article are included in the article's Creative Commonslicence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commonslicence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtainpermission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/.The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to thedata made available in this article, unless otherwise stated in a credit line to the data.

* Correspondence: MoreiraA@uthscsa.edu1Department of Pediatrics, Division of Neonatology, University of TexasHealth Science-San Antonio, San Antonio, TX 78229-3900, USAFull list of author information is available at the end of the article

Moreira et al. Stem Cell Research & Therapy (2020) 11:399 https://doi.org/10.1186/s13287-020-01900-7

BackgroundPulmonary illness is a leading cause of morbidity andmortality [1]. In children, acute respiratory exacerbationsare a common reason for primary care visits and areoften implicated in hospitalizations [2, 3]. Many of thesepulmonary conditions result in impairments in lungfunction that may last into adulthood [4, 5]. Conse-quently, identifying novel therapies for lung disease ishighly warranted.A unifying theme in many lung diseases includes in-

flammation [6–8]. While some inflammation is neces-sary to combat new disease and for proper woundhealing, chronic inflammation may result in altered lungstructure and function. During an acute illness, currenttherapies focus on restoring lung function by abating in-flammation [9–11]. For instance, glucocorticoids are themainstay therapy for reducing inflammation duringacute exacerbations of asthma [12]. More recently, mes-enchymal stromal/stem cells (MSCs) have shown en-couraging outcomes in animal models of lunginflammation [13–15].MSCs are promising agents as they are easily har-

vested, can be rapidly expanded, and can secrete factors(exosomes, microvesicles, microRNA) known to reduceinflammation [16–18]. The “secretome” or “conditionedmedia” of MSCs is considered biologically active and canbe easily collected from the surrounding fluid of propa-gating cells [19–21]. Remarkably, preclinical studies sug-gest MSC conditioned media (CdM) may be asrestorative as the MSCs themselves [22, 23]. We sup-ported this observation in a previous systematic reviewand meta-analysis demonstrating that CdM is as effect-ive as MSCs in modulating inflammation [24].This review is an extension of our previous work. In

this review, we examined the effects of CdM on (i) lungarchitecture/function in animal models recapitulatinglung disease and (ii) compare these findings to MSCs.Given that the therapeutic benefit of MSCs is attributedto a paracrine fashion, we believed CdM would havecomparable effects to MSCs.

MethodsOverview and literature searchThe methods in our review abide to those outlined bythe Systematic Review Centre for Laboratory Animal Ex-perimentation (SYRCLE) [25]. Our protocol was regis-tered through the Collaborative Approach to Meta-Analysis and Review of Data from Experimental Studies(CAMARADES) [26]. Details are described in our previ-ous publication.We conducted a literature search in five databases

using the following terms: mesenchymal stem cell-conditioned media, lung disease, and animal. The lastsearch was performed on March 17th, 2020. Three

independent investigators evaluated titles and abstracts,followed by full-text review.

Inclusion criteria and outcomes of interestWe included studies administering MSC-CdM to animalmodels of acute lung injury or acute respiratory distresssyndrome (ALI/ARDS), asthma, bronchopulmonary dys-plasia (BPD), chronic obstructive pulmonary disease(COPD), cystic fibrosis (CF), pneumonia, pulmonary fi-brosis (PF), and pulmonary hypertension (PH). Refer toSupplementary File 1 for the list of included studies.

Outcomes of interestMeasures of lung structure and/or function were ourprimary endpoint. Lung architecture and function wereassessed under the following categories: alveolarization,vasculogenesis, right ventricular hypertrophy, fibrosis,permeability, pulmonary pressures, compliance, and lunginjury. Although the pathogenesis of the included lungdiseases are heterogeneous, we combined all processesirrespective of disease. This was conducted to obtain ascoping overview of the impact of CdM on biologic pro-cesses implicated in lung disease. Subsequently, weassessed lung structure/function by disease in our sub-group analysis. Excluded studies were those which didnot provide data concerning our primary outcome ofinflammation.

Data extractionThree groups of investigators were used (ED and CE;RN and JM; ME, DM, and SM) to collect data. Uniform-ity of data was assessed by the primary author. This dataincluded general study design, animal model characteris-tics, conditioned media characteristics, and outcomes ofinterest.

Data analysisA random effects model was used to generate forestplots. A minimum of three studies were required foreach outcome to proceed with a meta-analysis. The esti-mated effect size of CdM or MSC on lung architecture/function was determined using standardized mean dif-ference (SMD) with a 95% confidence interval (CI). Stat-istical heterogeneity between studies was calculatedusing the I2 metric, and funnel plots were used to exam-ine publication bias. If more than six articles were in-cluded per outcome, we conducted a subgroup analysisfor disease, animal species, and route and dose of CdMadministration. All statistical analyses were performed inR version 3.6.2; packages used included dmetar, metafor,and meta.

Moreira et al. Stem Cell Research & Therapy (2020) 11:399 Page 2 of 15

Table

1Detailedsummaryof

inform

ationextractedfro

minclud

edstud

ies

No.

Autho

r(year)

Stud

yde

sign

Animalcharacteristics

Interven

tioncharacteristics

Outcomes

Disease

mod

elDisease

indu

ction

Animalmod

elGen

der

Age

Source;(Orig

in)

Dose;de

livery;

timing;

frequ

ency

Lung

architecture/functio

n

1Ahm

adi(2016)

Asthm

aOvalbum

inWistarrats

Male

Adu

ltBo

nemarrow

50μl;IV;1-daypo

stsensitizatio

n;×1

Trache

alreactivity

2Ahm

adi(2017)

Asthm

aOvalbum

inWistarrats

Male

Adu

ltBo

nemarrow

50μl;IT;1-daypo

stsensitizatio

n;×1

Histologiclung

injury

3Aslam

(2009)

BPD

Hyperoxia

FVBmice

Mixed

Neo

nate

Bone

marrow

50μl;IV;po

stnatal

day4;×1

Alveo

larization

RVH

Vasculog

enesis

4Chailakhyan(2014)

ALI

LPSfro

mE.coli

Wistarrats

Male

NR

Bone

marrow

1000

μl;IV;1hafter

LPSinjection;×1

Histologiclung

injury

5Chaub

ey(2018)

BPD

Hyperoxia

C57BL/6

mice

NR

Neo

nate

Hum

anum

bilical

cord

tissue

100μl;IP;PN

2and

PN4;×1

Alveo

larization

RVH

Pulm

onaryartery

pressure

6Cruz(2015)

Asthm

aAspergillus

fumigatus

sensitizatio

nC57/BL6

mice

Male

Adu

ltBo

nemarrow

200μl;IV;14

days

after

Aspergillus

challeng

e;×1

Histologiclung

injury

7Curley(2013)

ALI/

ARD

SHighstretch

mechanical

ventilatio

n

Spragu

e–Daw

ley

rats(patho

gen-fre

e)Male

Adu

ltBo

nemarrow

300μl;IT;2.5–3hpo

stinjury

initiation;×1

Alveo

larizationHistologic

lung

injury

Com

pliance

Wet,d

rylung

weigh

tratio

sBloo

dgas

8Felix

(2020)

PFBleomycin

Wistarrats

NR

Adu

ltAdipo

setissue

200μl;IV;10

days

after

indu

ction;×1

Histologiclung

injury

Fibrosis

9Gülaşı(2015)

BPD

Hyperoxia

Wistarrats

Mixed

Neo

nate

Bone

marrow

25μl;IT;on

the11th

day;

ateveryinspiratio

n;×1

Alveo

larization

10Hansm

ann(2012)

BPD

Hyperoxia

FVBmice

Mixed

Adu

ltBo

nemarrow

50μ l;IVpo

stnatald

ay14;×

1Alveo

larizationFibrosis

Com

pliance/Resistance

11Hayes

(2015)

VILI/ALI

Ventilator-indu

ced

Spragu

e–Daw

leyrats

Male

Adu

ltBo

nemarrow

500μl;1.5hafter

injury;×

1Alveo

larization

Perm

eability

Com

pliance

12Huh

(2011)

Emph

ysem

a(COPD

)Cigarette

smoke-

indu

ced

Lewisrats

Female

Adu

ltBo

nemarrow

300μl;IV;6mon

thsof

age;×10

Alveo

larization

Vascularization

Pulm

onaryartery

pressure

13Hwang(2016)

LIRI

Leftlung

was

clam

ped,

re-ven

tilated

,and

perfu

sed

Spragu

e–Daw

leyrats

Male

Adu

ltBo

nemarrow

200μl,IT,30

min

prior

todiseaseindu

ction;×1

Perm

eability

14Ione

scu(2012)

ARD

SLPSfro

mE.coli

C57/BL6

mice

Male

Adu

ltBo

nemarrow

30μl;IT;4hpo

st-LPS

expo

sure;×

1Perm

eabilityHistologic

lung

injury

15Kenn

elly(2016)

COPD

Receptor

knockout

NOD-SCID

IL-2rg

null

Mice

NR

NR

Hum

anbo

nemarrow

IN,d

ay0+6h;×2

Alveo

larization

16Keyhanmanesh(2018)

Asthm

aOvalbum

inWistarrats

Male

Adu

ltBo

nemarrow

50μl;IV,sing

ledo

se,

day33;rep

eateddo

seHistologiclung

injury

Moreira et al. Stem Cell Research & Therapy (2020) 11:399 Page 3 of 15

Table

1Detailedsummaryof

inform

ationextractedfro

minclud

edstud

ies(Con

tinued)

No.

Autho

r(year)

Stud

yde

sign

Animalcharacteristics

Interven

tioncharacteristics

Outcomes

Disease

mod

elDisease

indu

ction

Animalmod

elGen

der

Age

Source;(Orig

in)

Dose;de

livery;

timing;

frequ

ency

Lung

architecture/functio

n

days

33–35

17Li(2018)

PFSilica

Wistarrats

Female

Adu

ltBo

nemarrow

1mL,IT,d

ays1and4

post-silica;×

2Fibrosis

Histologiclung

injury

18Lu

(2015)

ARD

SLPSfro

mE.coli

C57/BL6

mice

Male

NR

Adipo

setissue

200μl;IV;4hpo

st-LPS

expo

sure;×

1Perm

eability

19Pierro

(2012)

BPD

Hyperoxia

New

born

rats

Mixed

Neo

nate

Hum

anum

bilical

cord

bloo

d7μl/g;IP;po

stnatald

ay4–21

(preventionstud

ies)

orfro

mpo

stnatald

ay14–28(re

gene

ratio

nstud

ies);×

18vs.×

15

Alveo

larization

Vascularization

RVH

Com

pliance

Exercise

capacity

20Rahb

argh

azi(2019)

Asthm

aOvalbum

inWistarrats

Male

Adu

ltBo

nemarrow

50μl;IT;day33;×

1Histologiclung

injury

21Rathinasabapathy

(2016)

PHMon

ocrotaline

Spragu

e–Daw

leyrats

Male

Adu

ltAdipo

setissue

100μl;IV;14

days

post-

MCTexpo

sure;×

1Vasculog

enesis

RVH

Fibrosis

22Sade

ghi(2019)

SMCEES

C57/BL6

mice

Male

6–8weeks

Adipo

setissue

500μl;IP;startweek

28;×

8Fibrosis

23Shen

(2014)

PFBleomycin

Wistarrats

Female

NR

Bone

marrow

200μl;IT;at

6handon

day3followingdisease

indu

ction;×2

Fibrosis

24Su

(2019)

ALI

LPSfro

mE.coli

C57BL/6

mice

Male

8–12

weeks

old

NR

200μl;IV;4hafter

diseaseindu

ction;×1

Lung

injury

25Sutsko

(2012)

BPD

Hyperoxia

Spragu

e–Daw

leyrats

Mixed

Neo

nate

Bone

marrow

50μl;IT;po

stnatal

day9;×1

Alveo

larization

Vascularization

RVH

26Trop

ea(2012)

BPD

Hyperoxia

FVBmice

NR

Neo

nate

Bone

marrow

50μl;IV;po

stnatal

day4;×1

Alveo

larization

27Wakayam

a(2015)

ARD

SBleomycin

C57/BL6Jmice

Female

Adu

ltHum

anexfoliated

decidu

ousteeth

500μl;IV;24

hpo

st-

bleo

mycin

expo

sure;×

1Fibrosis

28Waszak(2012)

BPD

Hyperoxia

Spragu

e–Daw

leyrats

Mixed

Neo

nate

Bone

marrow

1μl/g;IP;po

stnatald

ay0

topo

stnatald

ay21;×

22Alveo

larization

Vasculog

enesis

RVH

Pulm

onaryartery

pressure

29Zh

ao(2014)

Bron

chiolitisob

literans

Transplanted

dono

rtrache

aC57BL/6

mice

Male

Adu

ltPlacen

tade

rived

VolumeNR;IT;3rd

day

aftertransplantation;×1

Trache

alluminal

obliteration

ALIacutelung

injury,A

RDSacuterespira

tory

distress

synd

rome,

BPDbron

chop

ulmon

arydy

splasia,CE

ES-2

chloroeh

tyle

thylsulfide

,COPD

chronicob

structivepu

lmon

arydisease,

IPintrap

erito

neal,ITintratracheal,IV

intraven

ous,LIRI

lung

ischem

iarepe

rfusioninjury,LPS

lipop

olysaccharide,

MCT

mon

ocrotalin

e,NRno

trepo

rted

,PFpu

lmon

aryfib

rosis,RV

Hrig

htventricular

hype

rtroph

y,SM

sulfu

rmustard

chem

ical

lung

injury,V

ILI

ventilator-indu

cedlung

injury

Moreira et al. Stem Cell Research & Therapy (2020) 11:399 Page 4 of 15

ResultsStudy selectionOur literature search resulted in 245 articles. After re-moving duplicates and viewing the titles and abstracts,55 articles underwent full-text review. Twenty-nine arti-cles met inclusion (refer to Supplementary Figure 1).

Study detailsTable 1 summarizes the relevant study characteristics.Articles included in the review were published betweenthe years 2009 to 2020. BPD was the most common ani-mal model (n = 8), followed by ALI/ARDS (n = 5) andasthma (n = 5). All of the studies used rodents to inducetheir lung model.

CdM characteristicsConditioned media properties are summarized in Sup-plementary File 3. Stem cells were most isolated frombone marrows and cultured in Dulbecco’s modified Ea-gle’s medium. Incubation time of the CdM ranged from24 to 72 h. The volume of CdM administered rangedfrom 25 μl to 1 ml.

Alveolarization

� CdM: improved alveolarization with an SMD of 1.32(95% CI 0.99, 1.65; 12 studies; Fig. 1a) withmoderate heterogeneity (I2 = 67%; p < 0.01).

� MSC: improved alveolarization with an SMD of 1.80(95% CI 1.52, 2.07; 9 studies; Fig. 1b) with mildheterogeneity between groups (I2 = 36%; p = 0.01).

� CdM vs. MSC: no significant difference(Supplementary Figure 2).

Right ventricular hypertrophy

� CdM: favored CdM over control with an SMD of −1.08 (95% CI − 1.56, − 0.61); 6 studies; Fig. 2a) withsignificant heterogeneity (I2 = 70%; p < 0.01).

� MSC: favored over the control with an SMD of −1.05 (95% CI − 1.69, − 0.42; 3 studies, Fig. 2b) withsignificant heterogeneity between groups (I2 = 71%;p < 0.01).

� CdM vs. MSC: no significant difference (SMD −0.22, 95% CI − 0.36, 0.16; Supplementary Figure 3).

Lung fibrosis

� CdM: favored CdM over control with an SMD of −1.08 (95% CI − 1.56, − 0.61; 6 studies; Fig. 3a) withsignificant heterogeneity (I2 = 70%; p < 0.01).

� MSC: favored MSC over the control with an SMDof − 1.99 (95% CI − 2.93, − 1.04; 4 studies; Fig. 3b)

a

b

Fig. 1 Effect size of CdM (a) and MSC (b) on lung alveolarization.Forest plots demonstrate SMD with 95% confidence interval

Moreira et al. Stem Cell Research & Therapy (2020) 11:399 Page 5 of 15

with significant heterogeneity between groups (I2 =90%; p < 0.01).

� CdM vs. MSC: the comparison between CdMand MSCs was similar (refer to SupplementaryFigure 4).

Vasculogenesis

� CdM: superior to control with an SMD of − 2.46(95% CI − 3.22, − 1.70; 6 studies; Fig. 4a) withmoderate heterogeneity (I2 = 76%; p < 0.01).

� MSC: superior to control with an SMD of − 2.29(95% CI -3.01, − 1.56; 4 studies; Fig. 4b) with mildheterogeneity between groups (I2 = 35%; p = 0.14).

� CdM vs. MSC: overall effectiveness between CdMand MSCs again showed no significant difference(Supplementary Figure 5).

Permeability

� CdM: permeability assessment favored CdM overcontrol with an SMD of − 0.99 (95% CI − 1.32, −

a

b

Fig. 2 Effect size of CdM (a) and MSC (b) on right ventricular hypertrophy. Forest plots demonstrate SMD with 95% confidence interval

Moreira et al. Stem Cell Research & Therapy (2020) 11:399 Page 6 of 15

a

b

Fig. 3 Effect size of CdM (a) and MSC (b) on lung fibrosis. Forest plots demonstrate SMD with 95% confidence interval

Moreira et al. Stem Cell Research & Therapy (2020) 11:399 Page 7 of 15

0.66; 5 studies; Fig. 5a) homogeneity that is non-significant (I2 = 11.0%; p = 0.33).

� MSC: in the evaluation of permeability, the MSCwas favored over the control with an effect size of −1.54 (95% CI -2.13, − 0.95; 4 studies; Fig. 5b) withheterogeneity between groups (I2 = 57.0%; p < 0.01).

� CdM vs. MSC: equal effectiveness (SupplementaryFigure 6).

Pulmonary pressures

� CdM: improvement in right ventricular pressurescompared to control with an SMD of − 0.69 (95% CI− 0.99, − 0.39; 5 studies; Fig. 6a) with moderateheterogeneity (I2 = 51%; p < 0.01).

� MSC: superior to control with an SMD of − 1.63(95% CI − 2.02, − 1.24; 3 studies; Fig. 6b) withmoderate heterogeneity (I2 = 63%; p < 0.01).

� CdM vs. MSC: comparable (please refer toSupplementary Figure 7).

Histologic lung injury

� CdM: improvement in histologic lung injurycompared to control with an SMD of − 6.05 (95% CI− 8.72, − 3.38; 3 studies; Fig. 7a) with significantheterogeneity (I2 = 87%; p < 0.01).

� MSC: superior to control with an SMD of − 2.01(95% CI -3.41, − 0.60; 3 studies; Fig. 7b) withsignificant heterogeneity (I2 = 88%; p < 0.01).

� CdM vs. MSC: less than 3 studies; comparison notperformed.

Compliance

� CdM: improvement in lung compliance compared tocontrol with an SMD of 1.75 (95% CI 0.81, 2.69; 4

a

b

Fig. 4 Effect size of CdM (a) and MSC (b) on lung vascularization. Forest plots demonstrate SMD with 95% confidence interval

Moreira et al. Stem Cell Research & Therapy (2020) 11:399 Page 8 of 15

studies; Fig. 8a) with significant heterogeneity (I2 =76%; p < 0.01).

� MSC: improvement in lung compliance compared tocontrol with an SMD of 2.33 (95% CI 1.84, 2.82;3 studies; Fig. 8b) with no heterogeneity (I2 = 0%;p = 0.5).

� CdM vs. MSC: not applicable as less than threestudies performed a head-to-head comparison.

All outcomes for lung structure and function combined

� CdM: Supplementary Figure 8A shows the SMD of− 1.38 (with 95% CI of − 1.57, − 1.19) favoring CdMover control.

� MSC: Supplementary Figure 8B shows the SMD of− 1.66 (with 95% CI of − 1.91, − 1.41) favoring MSCover control.

� CdM vs. MSC: no difference was appreciatedbetween CdM and MSC when all outcomes werecombined (Supplementary Figure 8C).

Subgroup analysisStratification of data was performed by lung disease, tis-sue source, dose, and route of delivery of CdM. Evalu-ation was performed if more than 6 studies had data.

AlveolarizationSupplementary Figure 9A–D demonstrates that CdMhad the greatest impact on alveolarization in BPD

a

b

Fig. 5 Effect size of CdM (a) and MSC (b) on lung permeability. Forest plots demonstrate SMD with 95% confidence interval

Moreira et al. Stem Cell Research & Therapy (2020) 11:399 Page 9 of 15

a

b

Fig. 6 Effect size of CdM (a) and MSC (b) on pulmonary pressures. Forest plots demonstrate SMD with 95% confidence interval

Moreira et al. Stem Cell Research & Therapy (2020) 11:399 Page 10 of 15

animal models (SMD 1.67) and when the media wasderived from cord blood (SMD 2.89), given at a doseof 7 μl/g (SMD 2.89), and delivered via the intraperi-toneal route (SMD 1.56).

RVHSupplementary Figure 10A–D depicts that CdM sig-nificantly improved RVH in BPD animal models(SMD − 0.93) and only when the media was derivedfrom adipose tissue (SMD − 1.05), given at a doseof 100 μl (SMD − 1.14) and delivered intravenously(SMD − 0.86).

FibrosisSupplementary Figure 11A–D illustrates that CdM hadthe greatest impact in animal models of BPD and PH(SMD − 4.1, − 3.4, respectively) and when the media wasderived from adipose tissue (SMD − 2.61), given at adose of 50 μl (SMD − 4.10) and delivered intravenously(SMD − 1.95).

VascularizationSupplementary Figure 12A–D shows that CdM had thegreatest impact in animal models of COPD (SMD −8.09), when the media was derived from adipose tissue(SMD − 2.61), given at a dose of 300 μl (SMD − 8.09)and delivered intravenously (SMD − 3.65).

Risk of biasNo study was judged as low risk across all ten domains.Eight studies stated that the allocation selection was ran-dom. Most studies (n = 25) had similar groups at base-line. Risk of bias was large regarding allocationconcealment, whether authors mention random housingof animals, and blinding of caregivers or random selec-tion of outcome. All studies were found to sufficientlyreport complete data and being free from other bias.Refer to Supplementary File 2 [27].

Publication biasSupplementary Figures 13, 14, 15, 16, 17, 18, 19, and 20illustrate publication bias through funnel plots. Overall,

a

b

Fig. 7 Effect size of CdM (a) and MSC (b) on histologic lung injury. Forest plots demonstrate SMD with 95% confidence interval

Moreira et al. Stem Cell Research & Therapy (2020) 11:399 Page 11 of 15

publication bias was low in all the outcomes except forlung permeability.

DiscussionPreclinical studies reiterate the ability MSCs have ondampening lung inflammation. This capacity is largelydue to the paracrine secretion of MSC factors (microve-sicles, exosomes) that provide a basis for future cell-freetherapies for human disease [28–31]. This is the first re-view to directly compare the effects of CdM vs MSCs onlung structure and function in animal models of diverselung disease. Overall, we found that CdM improvedmeasures of alveolarization, right ventricular hyper-trophy, lung fibrosis, vasculogenesis and permeability.Furthermore, CdM reduced pulmonary pressures, ame-liorated histologic lung injury, and increased lung com-pliance. We found that CdM was comparable to MSCs

in all lung measures evaluated individually and whencombined.The bioactive factors contained in the CdM of MSCs

have been the focus of multiple studies and review arti-cles [32–34]. Congruent with the findings found in thisreview, Hansmann et al. show that MSC-CdM, com-pared to CdM from lung fibroblasts, reversed alveolarinjury, normalized lung function (airway resistance),and reversed RVH [35]. Additionally, the same group re-cently demonstrated that MSC exosomes (molecularcargo found within CdM) restored lung architecture,stimulated pulmonary blood vessel formation, and mod-ulated lung inflammation [22]. In an E. coli pneumonia-induced ALI mouse model, MSC microvesicles (alsofound in MSC-CdM) reduced lung permeability andhistologic injury score and were equivalent to MSCs[36]. Together, these findings, and those in recent

a

b

Fig. 8 Effect size of CdM (a) and MSC (b) on pulmonary compliance. Forest plots demonstrate SMD with 95% confidence interval

Moreira et al. Stem Cell Research & Therapy (2020) 11:399 Page 12 of 15

reviews, substantiate the results found in this review [37,38].This year, Augustine et al. published a network meta-

analysis comparing stem cell and cell-free therapies inpreclinical measures of BPD. MSC-CdM had a similareffect size to MSCs regarding alveolarization (MSC SMD1.71 vs. CdM SMD1.68), angiogenesis (SMD 2.24 vs.1.79), and pulmonary remodeling (1.29 vs. 1.22) [39].Similar to their results, this review showed that CdMhad among the largest impact on measures of alveolari-zation and vasculogenesis, processes critical for appro-priate lung healing, development, and function [40].Although vasculogenesis/angiogenesis is an importantprocess to restore lung function/structure, it can also en-hance remodeling and thus worsen outcomes in otherlung diseases such as asthma or pulmonary fibrosis [41].In Supplementary Figure 12A, we demonstrate that thisprocess improved in BPD, pulmonary hypertension, andCOPD but was not assessed in asthma/pulmonaryfibrosis.In the study by Hayes et al., they found that MSCs

were superior to CdM in a rodent model of ventilator-induced lung injury. However, our review suggests thatwhen you compile the literature, there were no signifi-cant benefits of using cells over CdM. We cannot ex-plain why CdM was not comparable in this study;however, an important challenge that remains in thefield includes the rigorous testing of key variables (tissuesource, dose, route, disease, etc.) that may impact thequality of CdM [42–44]. For instance, we found that theintravenous route provided optimal results. Moreover,multiple administrations of CdM may augment vasculardevelopment, as seen in the study by Huh et al (n = 10intravenous injections). Conversely, the optimal sourceand dose of CdM is dependent on the variable or the lungdisease. This brings to light that it will be incredibly chal-lenging to find a single CdM product that is ideal for alllung diseases. Thus, the idea of “one-size-fits-all” does nothold true for regenerative cells or products. Illustratingthis concept, Rathinasabapathy et al. showed greater im-provement in measures of RVH compared to other studiesmeasuring right ventricular size. Important differencesseen in the study by Rathinasabapathy and colleagues wasthat they used a different animal model (PH vs. BPD)and age of rodents (adult vs. neonatal) [45].As investigators, we should attempt to tease out these

characteristics in order to have the ideal product(s) forour lung disease of interest. In this way, we may havetranslational success in future clinical studies. Refiningthese features will take time but will play a vital role inefficacy. Moreover, pinpointing small and large animalmodels of lung disease that will recapitulate what occursat the patient bedside is essential if we want to move theneedle in the field [46].

The plausibility of using a cell-free product as atherapeutic agent for lung disease is substantiated bynewly registered human clinical trials. For instance,NCT04235296 and NCT04234750 are evaluatingsafety of MSC-CdM in regulating wound inflamma-tion and promoting wound healing in burn injury.Another Phase I trial (NCT04134676) plans to studythe therapeutic potential of umbilical cord tissue-derived stem cell CdM on chronic skin ulcers. Trialsvaluing the safety of stem cell CdM constituents (exo-somes) are also underway for ischemic stroke(NCT3384433) and ocular conditions (NCT04213248,NCT03437759).There are several limitations to our systematic review

and meta-analysis, many of which mirror those pub-lished in our previous report. We incorporated multipleanimal models of lung disease that have diverse patho-logic processes resulting in their etiology. Also, most ofthe studies lacked methodologic details rendering themwith an unclear risk of bias. Moreover, although preclin-ical models of lung disease have been helpful in identify-ing targetable mechanisms/processes, they oftentimeslack the intricacies of human disease. Thus, meticulousefficacy studies in large animals may be one approach tomitigate translational failure in human trials.

ConclusionThis review demonstrates that the administration ofCdM in animal models of lung disease improves lungarchitecture and function. When compared to MSCs,CdM is as efficacious and provides a basis that cell-freeproducts are a viable option for future studies. However,mores studies are needed to identify how specific vari-ables (tissue source, route of delivery, concentration,etc.) may impact/strengthen their therapeutic potential.

Supplementary informationSupplementary information accompanies this paper at https://doi.org/10.1186/s13287-020-01900-7.

Additional file 1: Figure S1. Flow diagram demonstrating studyselection process.

Additional file 2: Figure S2. Effect size of CdM vs. MSC on lungalveolarization. . Forest plots demonstrate SMD with 95% confidenceinterval.

Additional file 3: Figure S3. Effect size of CdM on right ventricularhypertrophy. Forest plots demonstrate SMD with 95% confidenceinterval.

Additional file 4: Figure S4. Effect size of MSC on lung fibrosis. Forestplots demonstrate SMD with 95% confidence interval.

Additional file 5: Figure S5. Effect size of CdM vs. MSC on pulmonaryvasculogenesis. Forest plots demonstrate SMD with 95% confidenceinterval.

Additional file 6: Figure S6. Effect size of CdM vs. MSC on lungpermeability. Forest plots demonstrate SMD with 95% confidenceinterval.

Moreira et al. Stem Cell Research & Therapy (2020) 11:399 Page 13 of 15

Additional file 7: Figure S7. Effect size of CdM vs. MSC on pulmonarypressures. Forest plots demonstrate SMD with 95% confidence interval.

Additional file 8: Figure S8. Effect size of CdM (a), MSCs (b), and CdMvs. MSC (c) on all eight outcomes. Forest plots demonstrate SMD with95% confidence interval.

Additional file 9: Figure S9. Effect size of CdM on lung alveolarizationby disease (a), source (b), dose (c), and route (d). Forest plotsdemonstrate SMD with 95% confidence interval.

Additional file 10: Figure S10. Effect size of CdM on right ventricularhypertrophy by disease (a), source (b), dose (c), and route (d). Forest plotsdemonstrate SMD with 95% confidence interval.

Additional file 11: Figure S11. Effect size of CdM on lung fibrosis bydisease (a), source (b), dose (c), and route (d). Forest plots demonstrateSMD with 95% confidence interval.

Additional file 12: Figure S12. Effect size of CdM on pulmonaryvascularization by disease (a), source (b), dose (c), and route (d). Forestplots demonstrate SMD with 95% confidence interval.

Additional file 13: Figure S13. Funnel plot assessing for publicationbias of CdM on lung alveolarization.

Additional file 14: Figure S14. Funnel plot assessing for publicationbias of CdM on right ventricular hypertrophy.

Additional file 15: Figure S15. Funnel plot assessing for publicationbias of CdM on lung fibrosis.

Additional file 16: Figure S16. Funnel plot assessing for publicationbias of CdM on pulmonary vasculogenesis.

Additional file 17: Figure S17. Funnel plot assessing for publicationbias of CdM on lung permeability.

Additional file 18: Figure S18. Funnel plot assessing for publicationbias of CdM on pulmonary pressures.

Additional file 19: Figure S19. Funnel plot assessing for publicationbias of CdM on histologic lung injury.

Additional file 20: Figure S20. Funnel plot assessing for publicationbias of CdM on lung compliance.

Additional file 21: File S1. List of articles included in this review.

Additional file 22: File S2. SYRCLE risk of bias.

Additional file 23: File S3. CdM characteristics.

AbbreviationsARDS: Acute respiratory distress syndrome; BPD: Bronchopulmonarydysplasia; CAMARADES: Collaborative Approach to Meta-Analysis and Reviewof Data from Experimental Studies; CdM: Conditioned media; CF: Cysticfibrosis; CI: Confidence interval; MSC: Mesenchymal stem/stromal cell;NCT: National clinical trial; PH: Pulmonary hypertension; SMD: Standardizedmean difference; SYRCLE: Systematic Review Centre for Laboratory AnimalExperimentation

AcknowledgementsDr. Robert Cote, Director of the Writing Skills Program at the University ofArizona, Tucson.

Authors’ contributionsAM contributed to the design of review, analysis of data, manuscript data,and oversight. RN contributed to the data collection, manuscript writing, andrisk of bias. KH contributed to the data collection and manuscript writing. CEcontributed to the database search, design of review, assembly of data, andanalysis of data. JM contributed to the data collection. AM contributed tothe design of review, manuscript writing, and interpretation of data. EDcontributed to the database search, design of review, and data collection. SZcontributed to the manuscript writing and interpretation of data. MEcontributed to the data collection, risk of bias, and Tables 1 and 2. DMcontributed to the data collection and Tables 1 and 2. SM contributed to thedata collection and Tables 1 and 2. The author(s) read and approved thefinal manuscript.

FundingSupported by NIH NCATS KL2 TR001118, UT Health San Antonio School ofMedicine pilot grant, Parker B. Francis Foundation (provided to seniorauthor).

Availability of data and materialsAvailability of data and materials will be available through figshare uponpublication of the manuscripts.

Ethics approval and consent to participateNot applicable.

Consent for publicationAll authors have looked through the manuscript and approved thesubmission.

Competing interestsThe authors declare that they have no competing interests.

Author details1Department of Pediatrics, Division of Neonatology, University of TexasHealth Science-San Antonio, San Antonio, TX 78229-3900, USA. 2Departmentof Pediatrics, Division of Critical Care, Baylor College of Medicine, Houston,TX, USA.

Received: 12 February 2020 Revised: 19 August 2020Accepted: 24 August 2020

References1. Ferkol T, Schraufnagel D. The global burden of respiratory disease. Ann Am

Thorac Soc. 2014;11:404–6. Available from: http://www.atsjournals.org/doi/abs/10.1513/AnnalsATS.201311-405PS. [cited 2019 Sep 30].

2. Winterstein AG, Choi Y, Cody MH. Association of age with risk ofhospitalization for respiratory syncytial virus in preterm infants with chroniclung disease. JAMA Pediatr. 2018;172:154–60.

3. Maxwell BG, Nies MK, Ajuba-Iwuji CC, Coulson JD, Romer LH. Trends inhospitalization for pediatric pulmonary hypertension. Pediatrics. 2015;136:241–50.

4. Bui DS, Lodge CJ, Burgess JA, Lowe AJ, Perret J, Bui MQ, et al. Childhoodpredictors of lung function trajectories and future COPD risk: a prospectivecohort study from the first to the sixth decade of life. Lancet Respir Med.2018;6:535–44.

5. Savran O, Ulrik CS. Early life insults as determinants of chronic obstructivepulmonary disease in adult life. Int J Chron Obstruct Pulmon Dis. 2018;13:683–93. https://doi.org/10.2147/COPD.S153555. Published 2018 Feb 26.

6. Zhou-Suckow Z, Duerr J, Hagner M, Agrawal R, Mall MA. Airway mucus,inflammation and remodeling: emerging links in the pathogenesis ofchronic lung diseases. Cell Tissue Res. 2017;367(3):537–50.

7. Virk H, Arthur G, Bradding P. Mast cells and their activation in lung disease.Transl Res. 2016;174:60–76.

8. Barnes PJ. Cellular and molecular mechanisms of asthma and COPD. Clin Sci(Lond). 2017;131(13):1541–58.

9. Robinson D, Humbert M, Buhl R, et al. Revisiting Type 2-high and Type 2-low airway inflammation in asthma: current knowledge and therapeuticimplications. Clin Exp Allergy. 2017;47(2):161–75.

10. Becerra-Díaz M, Wills-Karp M, Heller NM. New perspectives on theregulation of type II inflammation in asthma. F1000Research. 2017;6:1014.Available from: http://www.ncbi.nlm.nih.gov/pubmed/28721208. [cited 2019Jun 11].

11. Dias-Freitas F, Metelo-Coimbra C, Roncon-Albuquerque R Jr. Molecularmechanisms underlying hyperoxia acute lung injury. Respir Med. 2016;119:23–8.

12. Barsky EE, Giancola LM, Baxi SN, Gaffin JM. A Practical Approach to SevereAsthma in Children [published correction appears in Ann Am Thorac Soc.2018;15(6):767–8.

13. Curley GF, Hayes M, Ansari B, Shaw G, Ryan A, Barry F, et al. Mesenchymalstem cells enhance recovery and repair following ventilator-induced lunginjury in the rat. Thorax. 2012;67:496–501. Available from: http://www.ncbi.nlm.nih.gov/pubmed/22106021. [cited 2017 Dec 16].

14. Zhong H, Fan XL, Fang S, Bin LYD, Wen W, Fu QL. Human pluripotent stemcell-derived mesenchymal stem cells prevent chronic allergic airway

Moreira et al. Stem Cell Research & Therapy (2020) 11:399 Page 14 of 15

inflammation via TGF-β1-Smad2/Smad3 signaling pathway in mice. MolImmunol. 2019;109:51–7.

15. Cui P, Xin H, Yao Y, et al. Human amnion-derived mesenchymal stem cellsalleviate lung injury induced by white smoke inhalation in rats. Stem CellRes Ther. 2018;9(1):101.

16. Phinney DG, Pittenger MF. Concise review: MSC-derived exosomes for cellfree therapy. Stem Cells. 2017;35:851–8.

17. Vizoso FJ, Eiro N, Cid S, Schneider J, Perez-Fernandez R. Mesenchymal stemcell secretome: toward cell-free therapeutic strategies in regenerativemedicine. Int J Mol Sci. 2017;18 Available from: http://www.ncbi.nlm.nih.gov/pubmed/28841158. [cited 2017 Dec 16].

18. Moreira A, Kahlenberg S, Hornsby P. Therapeutic potential of mesenchymalstem cells for diabetes. J Mol Endocrinol. 2017;59:R109–20. Available from:http://www.ncbi.nlm.nih.gov/pubmed/28739632. [cited 2017 Nov 30].

19. Zakrzewski W, Dobrzyński M, Szymonowicz M, Rybak Z. Stem cells: past,present, and future. Stem Cell Res Ther. 2019;10(1):68.

20. Cruz FF, PRM R. Hypoxic preconditioning enhances mesenchymal stromalcell lung repair capacity. Stem Cell Res Ther. 2015;6:130. Available from:http://www.ncbi.nlm.nih.gov/pubmed/26169784. [cited 2017 Nov 16].

21. Ferreira JR, Teixeira GQ, Santos SG, Barbosa MA, Almeida-Porada G,Gonçalves RM. Mesenchymal stromal cell secretome: influencingtherapeutic potential by cellular pre-conditioning. Front Immunol. 2018;9:2837.

22. Willis GR, Fernandez-Gonzalez A, Anastas J, Vitali SH, Liu X, Ericsson M, et al.Mesenchymal stromal cell exosomes ameliorate experimentalbronchopulmonary dysplasia and restore lung function throughmacrophage immunomodulation. Am J Respir Crit Care Med. 2018;197:104–16. Available from: http://www.ncbi.nlm.nih.gov/pubmed/28853608. [cited2019 Jun 21].

23. van Haaften T, Byrne R, Bonnet S, Rochefort GY, Akabutu J, Bouchentouf M,et al. Airway delivery of mesenchymal stem cells prevents arrested alveolargrowth in neonatal lung injury in rats. Am J Respir Crit Care Med. 2009;180:1131–42. Available from: http://www.ncbi.nlm.nih.gov/pubmed/19713449.[cited 2016 Oct 27].

24. Emukah C, Dittmar E, Naqvi R, et al. Mesenchymal stromal cell conditionedmedia for lung disease: a systematic review and meta-analysis of preclinicalstudies. Respir Res. 2019;20(1):239.

25. Zeng X, Zhang Y, Kwong JS, et al. The methodological quality assessmenttools for preclinical and clinical studies, systematic review and metaanalysis,and clinical practice guideline: a systematic review. J Evid Based Med. 2015;8(1):2–10.

26. Welcome to CAMARADES. Available from: http://www.dcn.ed.ac.uk/camarades/. [cited 2020 Jan 30].

27. Hooijmans CR, Rovers MM, De Vries RBM, Leenaars M, Ritskes-hoitinga M,Langendam MW. SYRCLE’s risk of bias tool for animal studies. BMC Med ResMethodol. 2014;14:1–9.

28. Bruno S, Kholia S, Deregibus MC, Camussi G. The role of extracellularvesicles as paracrine effectors in stem cell-based therapies. Adv Exp MedBiol. 2019;1201:175–93.

29. Joo HS, Suh JH, Lee HJ, Bang ES, Lee JM. Current knowledge and futureperspectives on mesenchymal stem cell-derived exosomes as a newtherapeutic agent. Int J Mol Sci. 2020;21(3):727. Published 2020 Jan 22.https://doi.org/10.3390/ijms21030727.

30. Liu C, Wang J, Hu J, Fu B, Mao Z, Zhang H, et al. Extracellular vesicles foracute kidney injury in preclinical rodent models: a meta-analysis. Stem CellRes Ther. 2020;11:11.

31. Willis GR, Mitsialis SA, Kourembanas S. “Good things come in smallpackages”: application of exosome-based therapeutics in neonatal lunginjury. Pediatr Res. 2018;83:298–307. Available from: http://www.nature.com/articles/pr2017256. [cited 2019 Jul 12].

32. Madrigal M, Rao KS, Riordan NH. A review of therapeutic effects ofmesenchymal stem cell secretions and induction of secretory modificationby different culture methods. J Transl Med. 2014;12:260.

33. Pawitan JA. Prospect of stem cell conditioned medium in regenerativemedicine. Biomed Res Int. 2014;2014:965849. Available from: http://www.ncbi.nlm.nih.gov/pubmed/25530971. [cited 2019 Jul 20].

34. Augustine S, Avey MT, Harrison B, Locke T, Ghannad M, Moher D, et al.Mesenchymal stromal cell therapy in bronchopulmonary dysplasia:Systematic review and meta-analysis of preclinical studies. Stem Cells TranslMed. 2017;6:2079–93. Available from: http://www.ncbi.nlm.nih.gov/pubmed/29045045. [cited 2019 Mar 22].

35. Hansmann G, Fernandez-Gonzalez A, Aslam M, Vitali SH, Martin T, MitsialisSA, et al. Mesenchymal stem cell-mediated reversal of bronchopulmonarydysplasia and associated pulmonary hypertension. Pulm Circ. 2012;2:170–81.Available from: http://www.ncbi.nlm.nih.gov/pubmed/22837858. [cited 2018Jan 18].

36. Zhu YG, Feng XM, Abbott J, Fang XH, Hao Q, Monsel A, et al. Humanmesenchymal stem cell microvesicles for treatment of Escherichia coliendotoxin-induced acute lung injury in mice. Stem Cells. 2014;32:116–25.

37. Lanyu Z, Feilong H. Emerging role of extracellular vesicles in lung injury andinflammation. Biomed Pharmacother. 2019;113:108748.

38. Fujita Y, Kadota T, Araya J, Ochiya T, Kuwano K. Clinical application ofmesenchymal stem cell-derived extracellular vesicle-based therapeutics forinflammatory lung diseases. J Clin Med. 2018;7:355.

39. Augustine S, Cheng W, Avey MT, Chan ML, Lingappa SMC, Hutton B, et al.Are all stem cells equal? Systematic review, evidence map, and meta-analyses of preclinical stem cell-based therapies for bronchopulmonarydysplasia. Stem Cells Transl Med. 2020;9:158–68. Available from: https://onlinelibrary.wiley.com/doi/abs/10.1002/sctm.19-0193. [cited 2020 Jan 30].

40. Alvira CM. Aberrant pulmonary vascular growth and remodeling inbronchopulmonary dysplasia. Front Med. 2016;3:21. Available from: http://journal.frontiersin.org/Article/10.3389/fmed.2016.00021/abstract. [cited 2018Nov 8].

41. Kropski J, Richmond B, Gaskill C, Foronjy R, Majika S. Deregulatedangiogenesis in chronic lung diseases: a possible role for lungmesenchymal progenitor cells (2017 Grover Conference Series). Pulm Circ.2018;8:2045893217739807.

42. Sensebé L, Gadelorge M, Fleury-Cappellesso S. Production of mesenchymalstromal/stem cells according to good manufacturing practices: a review.Stem Cell Res Ther. 2013;4:66. Available from: http://www.ncbi.nlm.nih.gov/pubmed/23751270. [cited 2017 Jul 11].

43. Galipeau J, Sensébé L. Mesenchymal stromal cells: clinical challenges andtherapeutic opportunities. Cell Stem Cell. 2018;22(6):824–33.

44. Lopes-Pacheco M, Robba C, Rocco P, Pelosi P. Current understanding of thetherapeutic benefits of mesenchymal stem cells in acute respiratory distresssyndrome. Cell Biol Toxicol. 2020;36:83–102.

45. Rathinasabapathy A, Bruce E, Espejo A, et al. Therapeutic potential ofadipose stem cell-derived conditioned medium against pulmonaryhypertension and lung fibrosis. Br J Pharmacol. 2016;173(19):2859–79.https://doi.org/10.1111/bph.13562.

46. Chamuleau SAJ, van der Naald M, Climent AM, Kraaijeveld AO, Wever KE,Duncker DJ, et al. Translational research in cardiovascular repair. Circ Res.2018;122:310–8. Available from: http://www.ncbi.nlm.nih.gov/pubmed/29348252. [cited 2019 may 3].

Publisher’s NoteSpringer Nature remains neutral with regard to jurisdictional claims inpublished maps and institutional affiliations.

Moreira et al. Stem Cell Research & Therapy (2020) 11:399 Page 15 of 15